ABSTRACT: Melanoma is the deadliest form of skin cancer as patients presenting with metastatic disease have a five-year survival of only 2-16%. However, early clinical intervention prior to metastatic dissemination yields ten-year survival rates of ~92%. We aim to study how melanoma cells gain metastatic potential, hoping to expose future therapeutic avenues. The transcription factor, TWIST1 is known to be elevated in malignancies and play a role in metastatic progression; yet in melanoma little is known about TWIST1 regulated genes. Microarray analysis of the TWIST1 modulated transcriptome uncovered a negatively regulated adhesion molecule, CADM1. In other cancers, CADM1 has been shown to act as a tumor suppressor and is a co-stimulatory molecule for NK and CD8+ T-cells. How CADM1 functions in melanoma is largely unknown. Our preliminary data suggest that CADM1 suppresses melanoma invasion/migration, promotes anoikis, and is repressed at least in part by promoter methylation. Additionally, TCGA analysis demonstrates that patients with high CADM1 levels have a better response to immunotherapies. These data are the basis of this proposal which further examines how CADM1 functions in melanoma. The proposal will address the following specific aims, each having a mentored (K99) and an independent (R00) component: 1.) Identify domains and post-translational modifications of CADM1 that contribute to its anti-metastatic function. We will assay the ability of mutant CADM1 to promote anoikis, reduce invasion, and suppress extravasation in an in vivo model (K99), and analyze the downstream CADM1 effector proteins (R00). 2.) Identify the mechanism of TWIST1 mediated CADM1 regulation in melanoma. Tissue microarrays will be used to evaluate CADM1 expression as a function of disease stage, and we will define a role for TWIST1 in CADM1 promoter methylation (K99). We will also identify TWIST1 associated DNA methyltransferase(s) (R00). 3.) Determine if loss of CADM1 contributes to melanoma immune evasion. Melanoma cells with modulated expression of CADM1 will be subjected to in vitro cytotoxicity assays as well as in vivo tumor growth/tumor infiltrating lymphocyte assessments (K99). Furthermore, I will knock-down the putative CADM1 co-receptor (CRTAM) on T-cells, and test the efficacy of checkpoint blockade inhibitors against CADM1 modulated tumors (R00). Successful completion of these studies is linked to the training I will receive during the mentored phase of this award. To assist with this process, I have assembled a group of advisors led by mentor Dr. Andrew Aplin (Thomas Jefferson University - TJU), along with Drs. Meenhard Herlyn (Wistar Institute), Mauricio Reginato (Drexel University), Christopher Snyder (TJU), and Paolo Fortina (TJU). This group will help guide my research and will support my training efforts as part of my transition to an independent investigator. Completion of the goals in this proposal will define the function of an unknown metastatic suppressor in melanoma and provide me with the skills and training necessary to be a successful NIH funded independent investigator.